Pseudorabies virus is a herpes virus belonging to the genus alphaherpesvirinae. Although most warm-blooded species can serve as a host, either through natural or experimental means, this virus primarily resides within the swine population. In its active state, pseudorabies virus causes a disease that is generally fatal to young pigs. Those animals surviving infection become lifelong carriers, harboring the virus in an inactive, noninfectious state. The latent virus can be reactivated to its infectious state within these carriers and spread to other susceptible animals. This inactivation-reactivation cycle leads to perpetuation of the virus within a swine herd.
The polymerase chain reaction (PCR) method (Mullis and Faloona (1987) Meth. Enzymol. 155:335-350) provides for the enzymatic amplification of rare DNA sequences and/or minute quantities of DNA, enabling detection of rare DNA sequences not possible by other methods. The technique has been successfully utilized to detect a number of viral agents, such as human immunodeficiency virus (HIV) (Kwok et al. (1987) J. Virol. 61:1690-1694; Laure (1988) Lancet. 2:538-541; Murakawa et al. (1988) DNA 7:287-295; Ou et al. (1988) Science 239:295-297); human papillomavirus (Shihata (1988) J. Exp. Med. 167:224-230); HSV (Rowley et al. (1990) Lancet. 335:440-441); human rhinovirus (Gama et al. (1988) Nucleic Acids Res. 16(19):9346); hepatitis B (Larzul et al. (1988) J. Virol. Methods 20:227-237); human T-cell leukemia (Kwok et al. (1988) J. Infect. Dis. 158:1193-1197; Bhagavati et al. (1988) N. Engl. J. Med. 318:1141-1147); and pseudorabies virus (Maes et al. (1997) Vet. Microbiol. 55 (1-4): 13-27).
The PCR technique is currently the preferred method for detecting the presence of latent pseudorabies virus in animals in the absence of detectable infectious virus (Cheung (1995) Am. J. Vet. Res. 56(1):45-50). Initially this method was restricted to trigeminal ganglia tissues, which were believed to be the primary site of the latent virus. More recently, standard PCR and nested PCR methods have been used to demonstrate that tonsilar mucosal cells also harbor the latent virus (Cheung (1995) Am. J. Vet. Res. 56(l):45-50; Brown et al. (1995) Am J. Vet. Res. 56(5):587-594).
Detecting the latent virus in tonsilar tissues even with present PCR methods available in the art remains difficult. The frequency of viral DNA and RNA in tonsilar tissues is lower than that seen for trigeminal ganglion tissues. Yet use of tonsilar tissues is preferable, as suspected carriers of the virus do not have to be euthanized to obtain the sample.
Greater specificity for the latent pseudorabies virus is needed to enable more efficient detection, particularly in tonsilar tissues. To this end, the present invention provides for highly specific PCR primers that are used in an optimized nested polymerase chain reaction to detect a highly sensitive region of the pseudorabies virus gII gene.